Chronic psychosocial stress has been implicated in the etiology and progression of psychiatric disorders such as major depression and post-traumatic stress disorder (PTSD). In mice, we study the effects of psychosocial stress in a conflict paradigm that leads to behavioral alterations manifested as social avoidance, anhedonia, anxiety, and depressive-like states. In our chronic social defeat (CSD) paradigm, two male mice are placed in a continuous dyadic living relationship in which the subordinate experimental mouse is chronically exposed to and periodically defeated by a dominant mouse. Over the course of weeks in this living situation, the experimental mouse develops anxious and depressive-like behaviors that are associated with distinct neurochemical alterations in identified brain circuits. A decreased rate of hippocampal neurogenesis is one well-documented readout of CSD stress effects. Thus the paradigm is an ethologically relevant and validated model that can provide insights into the bases for mental illness in humans. We are beginning to address sex as a biological variable because psychiatric disorders show skewing of incidence by sex. We are currently adapting the CSD model to work in female mice, and we plan to evaluate the outcomes in both sexes. Our research focuses on the roles played by immune factors in the etiology of psychosocial stress-induced depressive states. We hypothesize that there is a bidirectional dialog between the brain and the immune system that serves to maintain homeostasis in healthy states, but disturbances within this dialog lead to homeostatic corrections or dysfunction that contribute to the onset and course of psychiatric disease. Therefore, we measure immune system parameters as we manipulate psychosocial processes that affect brain states. We have two projects that investigate immune changes in rodents undergoing psychological stress; one focuses on the role of the adaptive immune system in maintaining homeostatic balance during chronic stress, and the second investigates stress-induced changes in immune cells and molecules within the brain itself. 1) We conducted experiments aimed at addressing the role of the adaptive immune system in controlling mood states in the CSD model. The adaptive immune system, separate from the innate immune system, comprises lymphocytes that adapt toi.e., acquire and retain a memory ofprior challenges. We wondered whether lymphocytes might retain a memory of a stressful event, and if so, what effect might that might have on the brain and behavior. Lymphocytes normally reside in lymphoid organs and the blood, and they do not significantly cross the blood-brain barrier (BBB) to enter the brain. Surprisingly however, lymphocytes appear to exert effects on the brain and behavior as suggested by results of our lymphocyte transfer experiments. We published a novel approach to the question of adaptive immune system influence on the brain by studying the mutant Rag2-/- mouse, which has no adaptive immune system because it lacks a gene needed to produce mature lymphocytes. We transferred into this lymphopenic mouse lymphocytes from normal donor mice that had either been chronically stressed or unstressed, and after two weeks of lymphocyte reconstitution in the host mouse, behavioral tests and hippocampal cell proliferation assays were performed. The Rag2-/- mice that received cells from defeated mice showed anti-depressive and anxiolytic behaviors relative to mice that received no cell transfer or transfer of cells from home-cage control mice. These surprising findings suggested that the adaptive immune system retains a memory for adverse events and attempts to return the host animal to a condition of homeostasis. Current work examines the molecular and cellular determinants of the interaction and the anatomical and humoral pathways by which the immune system affects brain function and structure. We are particularly focused on the meninges as a site of interaction between the periphery and the brain. The meninges are a special component of the BBB because peripheral immune cellsleukocytesonly very sparsely enter the brain parenchyma but do reside in the subarachnoid spaces of the meninges, on the brain side of the BBB, where they can release bioactive molecules that circulate throughout the brain in the interstitial cerebrospinal fluid to potentially influence brain activity. We track leukocytes in the meninges using whole-mount meningeal preparations for histochemistry and flow cytometry for cell type analysis. We characterize activation states and gene expression profiles in these cells. Our analysis of cells in CSD stressed versus nonstressed animals has shown that stress alters the make-up of meningeal lymphocytes, skewing them in ways that suggest a pro-inflammatory phenotype. Similar studies are now directed at the meninges of Rag2-/- mice that received adoptive transfer. Pro- and anti-inflammatory lymphocyte phenotypes can be programmed in vitro, and thus parallel studies are underway to introduce programmed cells into Rag2-/- mice to determine whether they confer antidepressant properties. Such studies may lead to insights into new targets for therapeutic interventions in psychiatric disorders. 2) Specialized immune cells within the brain are called microglia, which share properties with peripheral immune cells of hematopoietic origin. Our previous published research documented effects of acute and chronic social defeat stress on microglial proliferation and activation states in the prefrontal cortex. These changes may be one basis for our published finding that chronically stressed mice had reduced levels of myelination in the prefrontal cortex. Stress-induced microglial activation likely alters neuronal function and thereby differentially direct behavioral outcomes associated with either susceptibility or resilience to the effects of the stress. We analyzed microglia isolated by flow cytometry to characterize cell-surface markers of activation and by microarray to assess gene expression patterns. The microarray data indicated that microglia isolated from defeated mice that had been susceptible (CSD-S) to the depressive effects of social defeat were functionally distinct from microglia taken from defeated mice that showed behavioral resilience (CSD-R) to the stress procedure. The microglial gene expression characteristics of the CSD-R group were more like those from unstressed home cage (HC) control mice, suggesting that microglial activity contributes to the brain states that afford resilience to the effects of psychosocial stress. Gene expression profiles in the microglia from CSD-S mice showed evidence of inflammation, phagocytosis, extracellular matrix breakdown, oxidative stress, and extravasation. The microarray findings were confirmed by histochemical and ex vivo assay methods. In the CSD-S mice only, local breeches of the BBB and brain micro-bleeds were found. CSD-S mice also showed elevated levels of dihydroethidium (DHE) in cells, indicating increased levels of oxidative stress. Drugs that prevent the conversion of microglia to a reactive pro-inflammatory state seen in the susceptible group may protect the animals from the depressive effects of the defeat stress. In one experiment, a reactive oxygen species (ROS) inhibitor N-acetyl cysteine protected against stress effects. Studies are underway to explore the role played by microglia in these effects by depleting microglia using the CSF-1R antagonist drug PLX5622. The aims of the work are to demonstrate a causal relationship between stress-induced immune alterations and susceptibility or resilience to the stress, and to suggest new therapeutic targets for the treatment of stress-related mood disorders.